Intestinal Organoids Research Articles

Fully synthetic hydrogels promote robust crypt formation in intestinal organoids.

Initial landmark studies in the design of synthetic hydrogels for intestinal organoid culture identified precise matrix requirements for differentiation, namely decompression of matrix-imposed forces and supplementation of laminin. But beyond stating the necessity of laminin, organoid-laminin interactions have gone largely unstudied, as this ubiquitous requirement of exogenous laminin hinders investigation. In this work, we exploit a fast stress relaxing, boronate ester based synthetic hydrogel for the culture of intestinal organoids, and fortuitously discover that unlike all other synthetic hydrogels to date, laminin does not need to be supplemented for crypt formation. This highly defined material provides a unique opportunity to investigate laminin-organoid interactions and how it influences crypt evolution and organoid function. Via fluorescent labeling of non-canonical amino acids, we further show that adaptable boronate ester bonds increase deposition of nascent proteins, including laminin. Collectively, these results advance the understanding of how mechanical and matricellular signaling influence intestinal organoid development.

Simple aneuploidy evades p53 surveillance and promotes niche factor-independent growth in human intestinal organoids.

Aneuploidy is nearly ubiquitous in tumor genomes, but the role of aneuploidy in the early stages of cancer evolution remains unclear. Here, by inducing heterogeneous aneuploidy in non-transformed human colon organoids (colonoids), we investigated how the effects of aneuploidy on cell growth and differentiation may promote malignant transformation. Previous work implicated p53 activation as a downstream response to aneuploidy induction. We found that simple aneuploidy, characterized by 1-3 gained or lost chromosomes, resulted in little or modest p53 activation and cell cycle arrest when compared with more complex aneuploid cells. Single-cell RNA sequencing analysis revealed that the degree of p53 activation was strongly correlated with karyotype complexity. Single-cell tracking showed that cells could continue to divide despite the observation of one to a few lagging chromosomes. Unexpectedly, colonoids with simple aneuploidy exhibited impaired differentiation after niche factor withdrawal. These findings demonstrate that simple aneuploid cells can escape p53 surveillance and may contribute to niche factor-independent growth of cancer-initiating colon stem cells.

Intestinal organoids to model Salmonella infection and its impact on progenitors

In order to survive and replicate, Salmonella has evolved mechanisms to gain access to intestinal epithelial cells of the crypt. However, the impact of Salmonella Typhimurium on stem cells and progenitors, which are responsible for the ability of the intestinal epithelium to renew and protect itself, remains unclear. Given that intestinal organoids growth is sustained by stem cells and progenitors activity, we have used this model to document the effects of Salmonella Typhimurium infection on epithelial proliferation and differentiation, and compared it to an in vivo model of Salmonella infection in mice. Among gut segments, the caecum was preferentially targeted by Salmonella. Analysis of infected crypts and organoids demonstrated increased length and size, respectively. mRNA transcription profiles of infected crypts and organoids pointed to upregulated EGFR-dependent signals, associated with a decrease in secretory cell lineage differentiation. To conclude, we show that organoids are suited to mimic the impact of Salmonella on stem cells and progenitors cells, carrying a great potential to drastically reduce the use of animals for scientific studies on that topic. In both models, the EGFR pathway, crucial to stem cells and progenitors proliferation and differentiation, is dysregulated by Salmonella, suggesting that repeated infections might have consequences on crypt integrity and further oncogenesis.

Personalized assessment of the effectiveness of targeted drugs for the treatment of cystic fibrosis in a patient with c.264_268delATATT and c.3139+1G>C rare variants of the <i>CFTR</i> gene

BACKGROUND: When new, most often rare CFTR gene variants, are detected in patients with cystic fibrosis, intestinal organoids are used, which allows for the assessment of the residual functional activity of the CFTR channel and the effect of targeted drugs to determine the possibility of further pathogenetic therapy. Clinical trials of the effectiveness of the targeted drugs in patients with rare CFTR variants are expensive, and patient groups are extremely small. A forskolin-induced swelling assay on a patient’s intestinal organoids allows for a personalized approach when studying rare or even single CFTR variants. AIM: To examine the effect of the CFTR potentiator ivacaftor and the combination of ivacaftor with the CFTR correctors lumacaftor, tezacaftor, and elexacaftor on the restoration of CFTR channel functions on a culture of intestinal organoids obtained from a patient with two rare CFTR variants c.264_268delATATT and c.3139+1GC. MATERIALS AND METHODS: To assess the activity of the CFTR channel, the forskolin-induced swelling assay on organoids and intestinal current measurements on rectal biopsy samples method were used. The clinical picture of a patient with the c.264_268delATATT/c.3139+1GC genotype was described. RESULTS: In vitro, the genetic variants c.264_268delATATT and c.3139+1GC lead to a complete loss of the functional CFTR protein, whereas CFTR modulators do not positively affect and do not lead to the restoration of CFTR function, in contrast from F508del/F508del control. The disease severity in a child is consistent with the results of functional tests. CONCLUSION: Both CFTR variants are classified as “severe” and cause a complete loss of chloride channel activity. No effective CFTR modulators were found for the c.264_268delATATT and c.3139+1GC variants; thus, targeted therapy cannot be recommended to the patient.

Human Milk Supports Robust Intestinal Organoid Growth, Differentiation, and Homeostatic Cytokine Production

Background and AimsNecrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using small intestinal organoids derived from fetal tissue of a gestational age similar to an extremely preterm infant, this study aims to assess the effect of diet on intestinal epithelial growth and differentiation to elucidate the role nutrition type plays in intestinal development and modifies the risk for NEC. MethodsOrganoids were cultured for 5 days in growth media and 5 days in differentiation media supplemented 1:40 with four different diets: parental milk (PM), donor human milk (DHM), standard formula, or extensively hydrolyzed formula. Images were captured daily and organoids were quantified. Organoids were preserved for RNA sequencing and immunofluorescence staining with Ki67, cleaved caspase 3, and chromogranin-A. Media was saved for cytokine/chemokine and growth factor analysis. ResultsHuman milk supplementation improved growth and differentiation of intestinal organoids generating larger organoids during the growth phase and organoids with longer and wider buds during differentiation compared to formula. Ki67 staining confirmed the proliferative nature of milk-supplemented organoids and chromogranin A staining proved that MM-supplemented organoids induced highest enteroendocrine differentiation. Human milk supplementation also upregulated genes involved in Wnt signaling and fatty acid metabolism pathways and promoted a homeostatic immune landscape, including via increased secretion of TNF-related apoptosis inducing ligand (TRAIL) among other cytokines. Conversely, organoids supplemented with formula had a downregulation of cell-cycle-promoting genes and a more inflammatory immune signature, including a reduced levels of leukemia inhibitory factor (LIF). ConclusionsOur results demonstrate that PM, and to a lesser extent DHM, support robust intestinal epithelial proliferation, differentiation, and homeostatic cytokine production, suggesting a critical role for factors enriched in human milk in intestinal epithelial health.

NLRP1B allele 2 does not respond to Val-boro-Pro (VbP) in intestinal epithelial cells

The intestinal mucosa must balance tolerance to commensal microbes and luminal antigens with rapid detection of enteric pathogens in order to maintain homeostasis. This balance is facilitated through the regulation of epithelial layer integrity by innate immune receptors. Certain NOD-like receptors (NLRs) expressed in intestinal epithelial cells, including NLRC4 and NLRP9B, form inflammasomes that protect against pathogens by activating caspase-1 to cause extrusion of infected cells. NLRP1B is a murine NLR encoded by five alleles of a highly polymorphic gene homologous to human NLRP1. NLRP1B forms inflammasomes in response to a variety of pathogens that cause intestinal infections, but it has almost exclusively been studied in immune cells and has not been characterized in cells of the intestinal epithelium. Here, we show that Nlrp1b allele 2 is expressed in ileal and colonic organoids derived for C57BL/6J mice, while the related gene Nlrp1a was not expressed. Nlrp1b was upregulated by interleukin-13 in organoids and by the protozoan Tritrichomonas muris in vivo, suggesting that NLRP1B may be involved in defense against enteric parasites. Surprisingly, while Val-boro-Pro (VbP) activated C57BL/6J-derived bone marrow-derived macrophages, which expressed both Nlrp1a and Nlrp1b, it did not activate intestinal organoids of the same genotype. We furthermore did not detect Nlrp1b in organoids derived from Balb/cJ mice, which express a different allele than the one expressed in C57BL/6J mice. Together, our results suggest that NLRP1B may have an allele-dependent function in murine IECs whose regulation is distinct from that of macrophages, and that the response to VbP might be exclusively driven by NLRP1A in C57BL/6J mice.

Astaxanthin Regulates Gene Expression in Mouse Small Intestinal Organoids in Culture

Astaxanthin Regulates Gene Expression in Mouse Small Intestinal Organoids in Culture

Assessment of P-glycoprotein function using canine intestinal organoid-derived epithelial interfaces

P-glycoprotein (P-gp), a multidrug efflux pump encoded by the ABCB1 (formerly MDR1) gene, plays a crucial role in limiting drug absorption and eliminating toxic compounds in both humans and dogs. However, species-specific differences in P-gp substrates necessitate the development of canine-specific evaluation systems. Canine intestinal organoids derived monolayers offer a promising platform for studying drug transport, yet P-gp-mediated transport in these models remains unexplored. We generated canine colonoid-derived 2D monolayers to investigate ABCB1 gene expression and P-gp function. We employed widely recognised P-gp substrates, Rhodamine 123 and Doxorubicin, in conjunction with the P-gp inhibitor PSC833 at Days 5 and 10 of culture. A significant increase in gene expression of P-gp encoded by the ABCB1 was noted on Day 10 compared to Day 5 of culture. Despite this disparity in gene expression, the transport activity of P-gp, as assessed by the efflux of Rhodamine 123 and Doxorubicin with PSC833 inhibition, did not exhibit significant differences between these two time points. However, the inhibition of P-gp function by PSC833 confirms the presence of functional P-gp in our model. Canine intestinal organoid-derived monolayers provide a valuable tool for investigating P-gp-mediated drug transport. These findings highlight the potential for predicting drug bioavailability and adverse reactions in veterinary medicine, aligning with principles of ethical and sustainable research.

Regulation of intestinal senescence during cholestatic liver disease modulates barrier function and liver disease progression

Background and AimsSenescence in cholangiocytes and hepatic stellate cells (HSC) has been described during human and murine cholestatic disease, having differential functions; detrimental in biliary cells and anti-fibrotic in HSC. Cholestatic liver disease associates with loss of intestinal barrier function and changes in the microbiome, the mechanistic cause of which is undetermined MethodsIntestinal samples were analysed from control and primary sclerosing cholangitis patients (PSC), wildtype (WT) and p16-3MR transgenic mice. Cholestatic liver disease was induced by bile duct ligation (BDL) and feeding with DDC diet. Fexaramine was used as an intestinal-restricted FXR agonist and antibiotics were given to eliminate the intestinal microbiome. Senescent cells were eliminated in p16-3MR mice with Ganciclovir and in WT mice with the senolytic drug ABT-263. In vitro studies were done in intestinal CaCo-2 cells and organoids were generated from intestinal-crypts isolated from mice. ResultsHere we show increased senescence in intestinal epithelial cells (IEC) in PSC patients and in mice after BDL and DDC diet. Intestinal senescence responded to reduced exposure to bile acids and increased presence of LPS in vitro and in vivo during cholestatic liver disease. Intestinal senescence associated with lower proliferation while increased intestinal stem cell (ISC) activation, as supported by increased organoid growth from ISC. Elimination of senescent cells with genetic and pharmacological approaches exacerbated liver injury and fibrosis during cholestatic liver disease that associated with increased IEC apoptosis and permeability. ConclusionsSenescence occurs in IEC during cholestatic disease and the elimination of senescent cells has a detrimental impact on the gut-liver axis. Our results point to cell-specific rather than systemic targeting of senescence as a therapeutic approach to treat cholestatic liver disease.

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Reprogramming human urine cells into intestinal organoids with long-term expansion ability and barrier function

Generation of intestinal organoids from human somatic cells by reprogramming would enable intestinal regeneration, disease modeling, and drug screening in a personalized pattern. Here, we report a direct reprogramming protocol for the generation of human urine cells induced intestinal organoids (U-iIOs) under a defined medium. U-iIOs expressed multiple intestinal specific genes and showed resembling gene expression profiles to primary small intestines. U-iIOs can be stably long-term expanded and further differentiated into more mature intestinal lineage cells with high expression of metallothionein and cytochrome P450 (CYP450) genes. These specific molecular features of U-iIOs differ from human pluripotent stem cells derived intestinal organoids (P-iIOs) and intestinal immortalized cell lines. Furthermore, U-iIOs exhibit intestinal barriers indicated by blocking FITC-dextran permeation and uptaking of the specific substrate rhodamine 123. Our study provides a novel platform for patient-specific intestinal organoid generation, which may lead to precision treatment of intestinal diseases and facilitate drug discovery.

The application of organoids in colorectal diseases.

Intestinal organoids are a three-dimensional cell culture model derived from colon or pluripotent stem cells. Intestinal organoids constructed in vitro strongly mimic the colon epithelium in cell composition, tissue architecture, and specific functions, replicating the colon epithelium in an in vitro culture environment. As an emerging biomedical technology, organoid technology has unique advantages over traditional two-dimensional culture in preserving parental gene expression and mutation, cell function, and biological characteristics. It has shown great potential in the research and treatment of colorectal diseases. Organoid technology has been widely applied in research on colorectal topics, including intestinal tumors, inflammatory bowel disease, infectious diarrhea, and intestinal injury regeneration. This review focuses on the application of organoid technology in colorectal diseases, including the basic principles and preparation methods of organoids, and explores the pathogenesis of and personalized treatment plans for various colorectal diseases to provide a valuable reference for organoid technology development and application.

Osteopontin Depletion in Nonhematopoietic Cells Improves Outcomes in Septic Mice by Enhancing Antimicrobial Peptide Production.

Osteopontin (Opn) depletion can improve septic outcomes, but the underlying mechanism remains unknown. In this study, we demonstrated that nonhematopoietic but not hematopoietic Opn depletion improved septic outcomes. When compared with wild type mice, cohoused Opn-/- mice displayed enhanced production of antibacterial peptides (AMPs), decreased bacterial loads, and a distinct bacterial composition of gut microbiota. Fecal microbiota transplantation and OPN neutralization assay showed that Opn depletion could reduce bacterial loads and improve septic inflammation. By employing an intestinal organoid culture system, we proved that OPN neutralization in wild type organoids could inactivate AKT and decrease FOXO3a phosphorylation, resulting in enhanced AMP production, whereas OPN treatment in OPN-deficient organoids could activate AKT and increase FOXO3a phosphorylation, leading to reduced AMP production. Our findings identified OPN as a novel regulatory factor of AMP production to modulate bacterial loads and composition of gut microbiota, in turn affecting sepsis outcomes.

Proinflammatory cytokines suppress stemness-related properties and expression of tight junction in canine intestinal organoids

Recent advancements in canine intestinal organoid research have paved the way for the development of enhanced in vitro models, crucial for exploring intestinal physiology and diseases. Despite these strides, there is a notable gap in creating specific in vitro models that focus on intestinal inflammation. Our study aims to bridge this gap by investigating the impact of proinflammatory cytokines on canine intestinal epithelial cells (IECs) within the context of organoid models. Canine intestinal organoids were treated with proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. The expression of stem cell markers Lgr5, Sox9, Hopx, and Olfm4 was evaluated through RT-qPCR, while membrane integrity was assessed using immunofluorescence staining for tight junction proteins and transport assays for permeability. IFN-γ significantly decreased Lgr5 expression, a key intestinal stem cell marker, at both 24 and 48 h post-treatment (p=0.030 and p=0.002, respectively). Conversely, TNF-α increased Olfm4 expression during the same intervals (p=0.018 and p=0.011, respectively). A reduction in EdU-positive cells, indicative of decreased cell proliferation, was observed following IFN-γ treatment. Additionally, a decrease in tight junction proteins E-cadherin and ZO-1 (p<0.001 and p=0.003, respectively) and increased permeability in IECs (p=0.012) were noted, particularly following treatment with IFN-γ. The study highlights the profound impact of proinflammatory cytokines on canine IECs, influencing both stem cell dynamics and membrane integrity. These insights shed light on the intricate cellular processes underlying inflammation in the gut and open avenues for more in-depth research into the long-term effects of inflammation on intestinal health.

The Role of Biophysical Factors in Organ Development: Insights from Current Organoid Models.

Biophysical factors play a fundamental role in human embryonic development. Traditional in vitro models of organogenesis focused on the biochemical environment and did not consider the effects of mechanical forces on developing tissue. While most human tissue has a Young's modulus in the low kilopascal range, the standard cell culture substrate, plasma-treated polystyrene, has a Young's modulus of 3 gigapascals, making it 10,000-100,000 times stiffer than native tissues. Modern in vitro approaches attempt to recapitulate the biophysical niche of native organs and have yielded more clinically relevant models of human tissues. Since Clevers' conception of intestinal organoids in 2009, the field has expanded rapidly, generating stem-cell derived structures, which are transcriptionally similar to fetal tissues, for nearly every organ system in the human body. For this reason, we conjecture that organoids will make their first clinical impact in fetal regenerative medicine as the structures generated ex vivo will better match native fetal tissues. Moreover, autologously sourced transplanted tissues would be able to grow with the developing embryo in a dynamic, fetal environment. As organoid technologies evolve, the resultant tissues will approach the structure and function of adult human organs and may help bridge the gap between preclinical drug candidates and clinically approved therapeutics. In this review, we discuss roles of tissue stiffness, viscoelasticity, and shear forces in organ formation and disease development, suggesting that these physical parameters should be further integrated into organoid models to improve their physiological relevance and therapeutic applicability. It also points to the mechanotransductive Hippo-YAP/TAZ signaling pathway as a key player in the interplay between extracellular matrix stiffness, cellular mechanics, and biochemical pathways. We conclude by highlighting how frontiers in physics can be applied to biology, for example, how quantum entanglement may be applied to better predict spontaneous DNA mutations. In the future, contemporary physical theories may be leveraged to better understand seemingly stochastic events during organogenesis.

Lactoferrin promotes intestinal stem cell‐mediated epithelial regeneration by activating Wnt signaling

AbstractNutritional intervention can greatly benefit people who suffer from side effects of medical treatments by improving intestinal post‐damage recovery. The recovery process is dependent on the regeneration of intestinal epithelial cells, which is driven by Lgr5+ intestinal stem cells (ISCs). Lactoferrin (LF) is a natural milk‐abundant protein with several gut health‐related functions such as antibacterial and immunoregulation activities, thus protecting intestine from damage. However, it is unclear whether LF also plays a role in the repair of the injured intestine, despite limited clues on its ability to modulate intestinal epithelial cell growth in vitro. Here, we show that LF accelerates intestinal epithelial recovery after both dextran sulfate sodium challenge on mice and TNF‐α treatment on intestinal organoids. Furthermore, we find LF gives rise to ISC‐mediated epithelial regeneration by directly activating the Lgr5+ stem cells. Finally, we identify Lrp5/Wnt signaling as the key pathway for LF‐enhancing ISC stemness and function. Overall, our study reveals the potential of LF as a nutrient that can be applied to promote intestinal healing.

A novel function of benzoic acid to enhance intestinal barrier defense against PEDV infection in Piglets

The intestinal barrier of newborn piglets is vulnerable and underdeveloped, making them susceptible to enteric virus infections. Benzoic acid (BA), employed as a growth promoter, exhibits the potential to enhance the gut health of piglets by modulating intestinal morphometry and tight junction dynamics. However, the extent to which BA regulates the intestinal mucus barrier through its impact on stem cells remains inadequately elucidated. Therefore, this study was conducted to investigate the effects of BA on the intestinal barrier and the differentiation of intestinal stem cells, employing in vivo piglet and in vitro intestinal organoid models. Our investigation revealed a significant increase in the number of goblet cells within the small intestine, as well as the strengthening of the mucus barrier in vivo following oral treatment with BA, providing partial protection against PEDV infection in piglets. Additionally, in vitro cultivation of enteroids with BA led to a notable increase in the number of MUC2+ GCs, indicating the promotion of GC differentiation by BA. Furthermore, transcriptome analysis revealed an upregulation of the number of GCs and the expression of cell vesicle transport-related genes during BA stimulation, accompanied by the downregulation of the Wnt and Notch signaling pathways. Mechanistically, MCT1 facilitated the transport of BA, subsequently activating the MAPK pathway to mediate GC differentiation. Overall, this study highlights a novel function for BA as a feed additive in enhancing the intestinal mucus barrier by promoting intestinal GC differentiation, and further prevents viral infection in piglets.

In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Deciphering HIV-associated inflammation: microbiome's influence and experimental insights.

To review novel experimental approaches for studying host:microbe interactions and their role in intestinal and systemic inflammation in people living with HIV (PLWH). Inflammation in PLWH is impacted by interactions between the microbiome, the intestinal epithelium, and immune cells. This complex interplay is not fully understood and requires a variety of analytical techniques to study. Using a multiomic systems biology approach provides hypothesis generating data on host:microbe interactions that can be used to guide further investigation. The direct interactions between host cells and microbes can be elucidated using peripheral blood mononuclear cells (PBMCs), lamina propria mononuclear cells (LPMC's) or human intestinal organoids (HIO). Additionally, the broader relationship between the host and the microbiome can be explored using animal models such as nonhuman primates and germ-free and double humanized mice. To explore complex host:microbe relationships, hypotheses are generated and investigations are guided by multiomic data, while causal components are identified using in-vitro and in-vivo assays.

Promoting Human Intestinal Organoid Formation and Stimulation Using Piezoelectric Nanofiber Matrices.

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology three days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.